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Departments of
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Neurology,
Molecular Microbiology and Immunology, and
Pathology, University of Southern California School of Medicine, Los Angeles, CA 90033; and
§
Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion and increased expression
of the early activation marker CD69, indicating differential regulation
of effector function. Although the total number of infiltrating T cells
declined following clearance of infectious virus, CD8+ T
cells, both specific for the dominant viral epitopes and of unknown
specificity, were retained within the CNS, suggesting an ongoing T cell
response during persistent CNS infection involving a virus-independent
component. Reversed immunodominance within the virus-specific
CD8+ T cell population further indicated epitope-specific
regulation, supporting ongoing T cell activation. Even in the absence
of infectious virus, the CNS thus provides an environment that
maintains both unspecific and Ag-specific CD8+ T cells with
restricted effector function. Chronic T cell stimulation may thus play
a role in preventing viral recrudescence, while increasing the risk of
pathological conditions, such as demyelination. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CNS is a preferred anatomical site for persistent viral infections (14, 15). The absence of classical lymphatic drainage, the presence of the blood brain barrier, low levels of MHC expression, and relative resistance of resident CNS cells to apoptosis are all factors contributing to inefficient immunity, thus providing a suitable environment for persistence (14, 16, 17). The mechanisms regulating the interactions between the immune response and the CNS as a target are poorly understood. Specifically, how the balance between viral replication, lymphocyte recruitment, and expression of effector function during acute infection contributes to the control of infection, viral persistence, and ultimately the severity of chronic disease has not been extensively explored. The recently discovered long-term presence of activated CD8+ T cells in the apparent absence of viral Ag or RNA (11) confirmed previous evidence that CD8+ T cells within the CNS are regulated differently compared with those in the periphery (10).
Intracerebral infection of rodents with the neurotropic JHM strain (JHMV)3 of mouse hepatitis virus (MHV) provides a paradigm for an acute CNS infection in its natural host resulting in persistence (18, 19). The acute infection, characterized by encephalitis associated with demyelination, is primarily controlled by CD8+ T cells infiltrating the parenchyma (20, 21). Infectious virus is typically cleared within 2–3 wk after infection, and mice eventually recover from hind limb paralysis (22). However, viral RNA (vRNA) can persist in the CNS up to 2 yr after clearance of infectious virus (23). Persistence is further characterized by ongoing primary demyelination (24). Both virus segregation to cells resistant to CTL-mediated lysis (20, 25) and vRNA sequence variability (23, 26, 27, 28) may contribute to the inability to provide sterile immunity. JHMV replicates preferentially in microglia, astrocytes, and oligodendrocytes, but only rarely in neurons (22, 29). During acute infection, the host immune response, specifically the CD8 component, clears virus from microglia and partially from astrocytes, and persisting RNA or Ag is predominantly detected in oligodendrocytes and astrocytes (25, 29, 30). Even following adoptive transfer of activated CD8+ T cells, oligodendrocytes remain relatively resistant to viral clearance; nevertheless, decreased demyelination suggests that potent early CD8+ T cell functions provide a more favorable clinical outcome (20). Although the peak incidence of CD8+ T cells coincides with a reduction in viral titers (31), CD8+ T cells are still found within the CNS of clinically recovered mice at 35 days postinfection (p.i.) (32). The role of virus-specific CD8+ T cells in the CNS during persistence is less clear, as very little is known about their functional state or specificity.
To begin to understand the relationship between viral replication and
CD8+ T cell regulation within the CNS, this study
characterizes CD8+ T cells throughout the acute
and persistent infection. Class I tetramer staining combined with
functional analysis revealed that up to 50% of
CD8+ T cells in the CNS of acutely infected mice
were virus-specific. Concomitant ex vivo cytolytic activity suggested
that persistence is not attributed to inadequate T cell recruitment or
expression of effector function. The CD8+
population in the CNS declined during the course of infection; however,
the percentage of tetramer+ cells remained high,
even after infectious virus was completely eliminated. IFN-
secretion was maintained during persistence. By contrast, Ag
responsiveness was impaired at the cytolytic level, indicating
differential regulation of distinct effector functions. Ongoing chronic
activation was further evident by a switch in immunodominance from
nucleocapsid (N) protein-specific CD8+ T cells
during the acute infection to spike (S) protein-specific
CD8+ T cells during persistence. Mechanisms
underlying the regulation of CD8+ T cells at the
initial site of infection may reside in increased CD69 expression and
delayed clearance of Ag from oligodendrocytes, compared with astrocytes
and macrophages. Although persisting CD8+ T cells
in the CNS may play a role in preventing viral recrudescence, chronic
immune stimulation and concomitant cytokine secretion may also
constitute a higher risk for immune-mediated pathogenesis than
previously appreciated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male CB6 F1 (H-2dxb) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 6 wk of age and certified naive to prior MHV exposure. Mice were housed in microisolator cages in an accredited animal facility at the University of Southern California (USC; Los Angeles, CA) and infected within 1 wk of arrival. Sublethal CNS infections were induced by intracranial injection of 32 µl containing 200 PFU of the 2.2v-1 mAb-derived variant of JHMV, as previously described (22). This variant produces paralysis associated with demyelination and replicates predominantly in oligodendrocytes. Viruses were propagated in the presence of neutralizing mAb J.2.2 and quantified by plaque assay using the murine delayed brain tumor (DBT) astrocytoma cell line, as described (29). For determination of tissue virus titers, one-half of the brain was homogenized in 2.0 ml of Dulbecco’s PBS (pH 7.4) using Tenbrock tissue homogenizers. Following centrifugation at 1500 x g for 7 min at 4°C, supernatants were assayed immediately or frozen at -70°C. Data presented are the average titers of individuals from groups of three or more mice.
Tissue sampling and isolation of lymphocytes
CNS mononuclear cells were obtained from pooled brains and spinal cords (SC) of 6–10 mice per group at various time points p.i., as described (32). Briefly, tissues were minced and homogenized in Tenbrock homogenizers. Cells were adjusted to 30% Percoll (Pharmacia, Uppsala, Sweden) and pelleted onto a 70% Percoll cushion, washed, and resuspended in RPMI medium. Typical yields were 0.8–2.0 x 106 cells per mouse, depending upon the disease state. Single cell suspensions were prepared from the spleens and cervical lymph nodes (CLN) from identical groups of mice, as previously described (33).
CTL assays and synthetic peptides
CTL assays were performed as described (34). Briefly, J774.1 (H-2d) or EL-4 (H-2b) target cells were labeled with 100 µCi Na51CrO4 (New England Nuclear, Boston, MA) and peptides added to washed target cells at a final concentration of 1 µM before addition of CTL at the indicated E:T ratios. After 4 h of incubation, 100 µl supernatant was removed and specific 51Cr release determined. Specific lysis was defined as: 100 x [(experimental release) - (spontaneous release)]/[(detergent release - (spontaneous release)]. Maximum spontaneous release values were <15% of the total release values in all experiments.
The pN318 (APTAGAFFF) and pS510 (CSLWNGPHL) peptides (34, 35, 36) were synthesized by the USC Norris Cancer Center Microchemistry Laboratory and purity assessed by HPLC and mass spectrometry. For T cell assays, peptides were solubilized at 1 mM in DMSO and diluted in PBS, as described (34).
Enzyme-linked immunospot (ELISPOT) assays
ELISPOT assays to measure the frequency of Ag-specific
IFN--secreting cells were conducted as described (32, 37). Serial 2.5-fold dilutions of cells were plated in
triplicate into 96-well plates supporting cellulose ester membranes
(MultiScreen HA; Millipore, Bedford, MA) precoated with R4-6A2 mAb
(PharMingen, San Diego, CA) and stimulated in the presence of
irradiated (25 Gy) splenocytes from naive mice (5 x
105/well), either pulsed with 1 µM pS510 or
pN318 peptide or left untreated. EL-4 supernatant was added as a source
of IL-2 to a final 2.5%, and cultures were incubated for 36 h at
37°C. Bound IFN- was detected by overnight incubation at 4°C
with biotinylated anti-IFN- mAb (0.5 µg/ml, XMG1.2;
PharMingen), followed by consecutive incubations with
streptavidin/peroxidase (Sigma, St. Louis, MO) and
3,3'-diaminobenzidine as substrate (Sigma).
Ld-N318 and Db-S510 tetramers
MHC class I heavy chains Ld and Db associated with viral 9-mer peptides pN318 and pS510, respectively, were generated as previously described (4). Monomers were converted to tetramers using either allophycocyanin (APC)-labeled streptavidin or R-PE-labeled Neutra-Lite avidin (Molecular Probes, Eugene, OR) at a molar ratio of 4:1.
Flow cytometry
Single cell suspensions were blocked with purified anti-mouse CD16/CD32 (2.4G2; PharMingen, San Diego, CA). For two- or three-color flow cytometric analysis, cells were stained with PE- or APC-conjugated tetramers (0.1–0.2 µg/0.5–1.0 x 106 cells) and various combinations of FITC- or PE-conjugated mAb specific for CD8 (53-6.7), CD4 (GK1.5), CD62L (MEL-14), CD44 (IM7), CD69 (H1.2F3), CD25 (PC61), CD11a (2D7), CD49d (R1-2) (all from PharMingen). Cells were stained at 4°C with mAb for 15–20 min before incubation with tetramers for 30 min in PBS containing 0.1% BSA. Samples were analyzed by flow cytometry on a FACStar (Becton Dickinson, Mountain View, CA). Three-color flow cytometry was performed on a Coulter (Hialeah, FL) Elite ESP equipped with an Argon-ion laser emitting 488 nm and a Helium-neon laser emitting 633 nm. Detection of FITC was performed at 520 nm emission using a 525 ± 5 nm bandpass filter, PE at 578 nm emission using a 575 ± 20 nm bandpass filter, and APC at 661 nm emission using a 675 ± 5 nm bandpass filter. Forward and side scatter signals obtained in linear mode were used to establish a gate that contained intact lymphocytes, while excluding remaining tissue debris. A minimum of 5 x 105 viable cells was stained and 5 x 104 to 1 x 105 events per sample analyzed.
RNA extraction and RT-PCR analysis
One-half of the brain or SC per mouse was processed individually for RNA isolation as previously described (26). Synthesis of cDNA was conducted by RT using 2 µg total RNA and 1 µg random primer, as previously described (26), and amplified using primers spanning N gene nucleotides 825-1227 (JN825, GAT GCC GGC CAG CCT AAG C; JN1227, CTA TAC ATC TCG ACC ACC ATC TTG) and S gene nucleotides 1390–1895 (JS1390, GAT GTT GCC TAC GCC CAG C; JS1895, GTT AAC CAT AGA GGT CAT ATC TGA CGC), respectively. Oligonucleotides were purchased from Genosys Biotechnologies (The Woodlands, TX). PCR amplification was conducted in 30 cycles using 20–50% of the RT reaction as described (26). Semiquantitative determination of relative vRNA levels was conducted by simultaneously probing serial dilutions of PCR products obtained from all time points with 32P-labeled internal oligonucleotides (JN984, AGC CAT GGT AAA GCT TGG AAC TAG TGA TCC; JS1642, GCC CAC AGC GGA GAT CAT AC). Radioactive signals were quantitated and normalized to signals for the housekeeping enzyme hypoxanthine phosphoriboxyltransferase (HPRT) to adjust for variable amounts of cDNA (38). The sample with the highest signal ratio of viral cDNA to HPRT cDNA was designated as maximal and arbitrarily set to 1 for each cDNA species analyzed; values for remaining samples were derived as percentages of the maximal ratio obtained for each distinct viral cDNA. Data shown are mean values for three to four mice per time point.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Infection of mice with the JHMV variant 2.2v-1 produces an acute,
nonfatal encephalomyelitis with extensive demyelination (22, 39). CD8+ T cells comprise the major
antiviral effectors by reducing virus replication in astrocytes and
microglia via a perforin-dependent mechanism and in oligodendroglia via
cytokine secretion (25, 29). C57BL/6
(H-2b) mice mount a dominant CTL response to a
Db-restricted epitope (S510) contained within the
viral S protein (35, 36), while BALB/c
(H-2d) mice respond predominantly to an
Ld-restricted epitope in the viral N protein
(N318) (33, 34). CB6 F1
(H-2dxb) mice were chosen to examine the
expansion and fate of CD8+ T cell populations
specific for the abundant and conserved N protein (40),
compared with the less abundant, hypervariable S protein (28, 41). Following infection of CB6 F1 mice,
virus titers in the brain declined to undetectable levels by day
16 p.i. (Fig. 1
), similar to
parental C57BL/6 and BALB/c mice (38). All mice developed
clinical symptoms with varying severity by day 12 and partially
recovered from hind limb paralysis by day 21. Few mice showed minor
residual signs of paralysis by day 45 p.i., the last time point
analyzed.
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Mononuclear cells isolated from the CNS of acutely JHMV-infected
BALB/c or C57BL/6 mice exhibit ex vivo cytolytic activity. By contrast,
lymphocytes from the spleen or CLN require in vitro stimulation to
detect cytolytic function (33, 42). Priming of
CD8+ T cells specific for both epitopes and
recruitment into the CNS was confirmed in acutely infected CB6
F1 mice (Fig. 2A). Mononuclear cells from
the CNS demonstrated similar cytolytic activities specific for both the
S510 and N318 epitopes, whereas no ex vivo cytolytic activity was
recovered from either the spleen or CLN. These data support previous
suggestions that JHMV-specific CTL rapidly accumulate to high numbers
within the CNS and/or acquire Ag-driven effector function at the site
of viral replication (33, 42). To examine whether ex vivo
cytolysis reflects a higher proportion of Ag-specific
CD8+ T cells in the CNS vs periphery,
CD8+ T cells localizing to the CNS during acute
infection were compared with those present in the peripheral lymphoid
organs. Mononuclear cells were stained for both CD8 and either
Db-S510- or
Ld-N318-specific TCRs using class I tetramers
(4, 43). At day 8 p.i., 
40% of CNS-derived
CD8+ T cells were virus-specific, with 26%
specific for the N epitope and 13% specific for the S epitope (Fig. 2B). By contrast, neither CD8+ T cells
present in the spleen nor CLN exhibited tetramer staining above 1%. Ex
vivo cytolytic effector function of CNS-derived T cells therefore
directly coincided with a high frequency of Ag-specific T cells within
the CNS compared with the periphery.
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Clearance of infectious virus is generally associated with a
severe decline in Ag-experienced T cells concomitant with entry into a
quiescent memory phase (1). However, data obtained from
immune mice challenged with a neurotropic influenza virus suggest that
virus-specific CD8+ T cells may be maintained in
the CNS in a chronically activated, yet nondividing state for extended
periods of time in the absence of detectable vRNA (11).
JHMV RNA persists in the CNS for up to 2 yr p.i. (23),
suggesting that low levels of Ag presentation may contribute to chronic
CD8+ T cell activation. Analysis of persistently
infected CB6 F1 mice, which had cleared
infectious virus, indeed confirmed retention and/or ongoing recruitment
of virus-specific CD8+ T cells in the CNS.
CNS-derived mononuclear cells contained a significant proportion
(8–12%) of CD8+ T cells at day 45 p.i.
(Fig. 3A). Furthermore,
tetramer staining revealed a slight increase in virus-specific
CD8+ T cells (48%) compared with days 8 and
12 p.i. (39%), indicating that both virus-specific and
nonspecific CD8+ T cells persist in the CNS.
Although the total percentage of virus-specific
CD8+ T cells remained similar, the pattern of
immunodominance had reversed comparing the acute and persistent
infections. Whereas the N-specific population decreased from 26% to
18%, S-specific CD8+ T cells increased from 13%
to 30%. Similar to the acute phase, peripheral virus-specific
CD8+ T cells remained at detection thresholds of
tetramer staining (data not shown).
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B). Correlation of E:T ratios with
tetramer+ populations suggested that the loss of
cytolytic function was not due to decreased numbers of virus-specific
CD8+ T cells in the effector population (Fig. 3B). Furthermore, CD4+ T cells
required to maintain CD8 effector function during viral clearance
(21) remained present in the CNS at similar percentages as
CD8+ T cells throughout the infection (data not
shown). Although loss of cytolytic activity may comprise a regulatory
mechanism to minimize tissue damage, the contribution of persisting
vRNA, leading to Ag expression and chronic T cell stimulation, or of
other local suppressive factors in inducing this anergic state, is
unclear (10, 44).
Phenotypic comparisons of CD8+ T cells from the
CNS during acute and persistent infection revealed similar expression
patterns of activation/memory markers. The majority of CNS-infiltrating
CD8+ T cells during acute infection were
CD44high (93.5%) and
CD62low (82.8%), CD11a
high (87.9%) and CD49d+
(79.4%), characteristic of an activated/memory phenotype
(1). Furthermore, as only 40–50% of the
CD8+ T cells were
tetramer+, these markers did not discriminate
virus-specific from nonspecific CD8+ T cells.
However, reduction of infectious virus was associated with an increased
frequency of CD69+ CD8+ T
cells, revealing a distinct phenotypic difference between
CD8+ T cells isolated early and late during
infection (Fig. 4). In contrast to the
other activation markers examined, the early activation Ag CD69 was
consistently expressed on <55% of CD8+ T cells
recovered on day 8 p.i. Although this implicated CD69 as a
potential marker to distinguish the virus-specific population,
examination of both N- and S-specific TCR subsets using three-color
flow cytometry revealed CD69 expression on both the
tetramer+ and tetramer-
populations (Fig. 4). Thus, the majority (55–70%), but not all, of
tetramer+ CD8+ T cells
expressed CD69 at day 8 p. i., with a slightly higher percentage
within the N-specific population (Fig. 4B). A marked
increase in CD69+ expression to almost 100%
occurred on both tetramer+ and
tetramer- CD8+ populations
between day 8 and 16 p.i., coincident with clearance of infectious
virus from the CNS. Thereafter, the numbers of
CD69+ T cells declined, but remained constantly
elevated at 90% in both the total CD8+
population and the tetramer+ subsets. Although
the CD69+ CD8+ T cell
population likely comprises cells recently stimulated within the CNS,
only a comparatively low percentage of CD8+ T
cells expressed the IL-2R 
-chain, even at day 8 p.i. (<1.0%),
consistent with phenotypic analysis of CD8+ T
cells from the CNS of JHMV-infected rats (45).
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An inverse frequency of epitope-specific
CD8+ T cell populations during the transition
from acute infection to persistence suggested a distinct Ag-driven
turnover process of N- vs S-specific CD8+ T cells
(see Fig. 3 above). Examination of the frequency of N- and S-specific
CD8+ T cells within the CNS during the course of
infection revealed that the transition coincided with the clearance of
infectious virus (Fig. 5). Initially
N-specific CD8+ T cells dominated up to day
12 p.i. Between days 14 and 21, this population declined,
concomitant with an increase in S-specific CD8+ T
cells. The transition exhibited fluctuating percentages within
different groups of mice (Fig. 5); however, the final phase after day
43 p.i. was characterized by a consistent prevalence of S-specific
CD8+ T cells.
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B) and similar
potency during acute infection (Fig. 2A) showed that altered
epitope hierarchy was not evident at the cytolytic level. To relate the
altered pattern of immunodominance to an independent immune function,
the frequency of IFN--responsive cells specific for each epitope was
determined by ELISPOT (Fig. 6). A high
frequency of T cells responding to both CTL epitopes was observed at
day 8 p.i. At this time, N-responsive cells prevailed over
S-responsive cells, consistent with the phenotypic immunodominance
demonstrated by tetramer staining. Although the frequencies of both
subsets declined markedly by days 12 and 16 p.i., the N-responsive
population remained dominant at day 12 and declined to the frequency of
S-responsive cells at day 16. By contrast, an increased number of
IFN- secreting S-specific T cells was found at day 21 p.i., at
which time phenotypic staining was unable to distinguish an altered
frequency of Ag-specific CD8+ T cell subsets
(Fig. 5). The overall higher frequency of IFN--secreting cells at
day 21 compared with day 16 p.i., suggested the possibility of a
second wave of infiltrating virus-specific T cells. The dominance of
S-specific CD8+ T cells, compared with the acute
phase of infection, was maintained to day 45 p.i. Overall, the
inverse ratios of S- vs N-specific IFN--secreting cells confirmed
the phenotypic difference at the functional level. Importantly, the
total frequency of JHMV-specific IFN--secreting cells was only
slightly lower during persistence at day 45 p.i. compared with the
acute phase at day 8 p.i., despite a <2-fold drop in
CD8+ T cells. Furthermore, cells isolated from
the CNS at day 21 p.i. showed a 2-fold increase in the frequency
of S- over N-specific cells, despite evidence of codominant populations
by phenotypic analysis (see Fig. 5). These data indicated that the
transition in immunodominance was evident earlier at a functional level
compared with phenotypic analysis.
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A). However, at day
8 p.i., vRNAs decreased in the brain and to a lesser extent in the
SC. Only one mouse had prominent levels of N gene sequences in the
brain, although the identical sequence was readily detected in the SC
of all four animals analyzed. By contrast, S gene sequences were only
found in the SC of a single mouse at day 8 p.i. At days 12 and
14 p.i., RNAs encoding both the N and S genes were found
predominantly within the SC. Whereas RNA containing N gene sequences
was still detected in the SC at day 21 p.i., S gene sequences
dropped to below detection. At day 45 p.i., both gene sequences
had dropped below detection levels, although increased amplification
confirmed their persistence (data not shown). As mutations in the S
epitope (27), but not N epitope (26), play a
role in JHMV pathogenesis, dominance of S-specific
CD8+ T cells within the CNS due to epitope
mutations, thereby preempting activation-induced cell death, was
examined. However, sequence analysis of S gene-specific PCR products
derived from the CNS of four individual mice on day 45 p.i.
revealed no evidence for mutations within the S epitope (data not
shown). CTL escape mutations therefore do not account for preferential
survival of S-specific CD8+ T cells during
persistence. Relative to HPRT encoding RNA, vRNA within the SC peaked
at day 8 and 12 p. i.; RNA encoding the N protein was prominent at
both days, compared with delayed detection of S protein encoding RNA
(Fig. 7B), likely reflecting the predominance of N gene
sequences in the nested set of subgenomic RNAs expressed during the
replication cycle (40). These data suggest that the rapid
accumulation and subsequent loss of N-specific
CD8+ T cells may reflect an increased
susceptibility of this T cell subset to activation-induced apoptosis
(46).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Potent local effector function associated with a high frequency of
tetramer+ T cells has also been demonstrated in
other nonlymphoid tissues such as the lung (3). A further
characteristic of tissue-restricted infections is the paucity of
Ag-specific CD8+ T cells in peripheral lymphoid
organs. During primary influenza virus infection,
CD8+ T cells within the lung contain 20%
virus-specific T cells, compared with only 1–2% in spleen and
regional nodes (3). By contrast, during primary
lymphocytic choriomeningitis virus (LCMV) infection, >50% of splenic
CD8+ T cells are virus-specific (4).
However, unlike influenza virus and JHMV, productive LCMV replication
in secondary lymphoid organs may contribute to the potent splenic
response. Localized infections in nonlymphoid organs thus recruit
virus-specific T cells into the infected tissue, giving rise to
effector function concentrated at the site of Ag. The high frequency of
tetramer+ CD8+ T cells
within the JHMV-infected CNS further suggests highly efficient Ag
presentation, despite relatively low levels of class I expression
(16, 50).
Following clearance of infectious virus, a significant population of CD8+ T cells is retained within the CNS. Furthermore, the percentage of tetramer+ cells did not decline. This contrasts with nonpersistent infections in which the vast majority of Ag-experienced CD8+ T cells undergo apoptosis following virus clearance, leaving a small population of quiescent memory cells and little evidence for retention of virus-specific cells at the previous site of infection (1, 3, 4). Similar to our results, tetramer+ cells are maintained at relatively high percentages following recovery of acute EBV infection, evoking viral persistence as a driving force (8). The degree to which viral persistence or the unique anatomic properties of the CNS drive T cell maintenance during persistent JHMV infection is unclear. RNA, but not infectious JHMV, can be detected 2 yr p.i. (23). However, nonproliferating CD8+ T cells may reside in the CNS even in the absence of viral persistence (11). This contrasts with the regulation of CD4+ T cells, which efflux from the CNS in the absence of cognate Ag recognition (51, 52, 53). Persisting CD8+ T cells within the CNS may result from trapping or ongoing recruitment from the periphery. The latter would involve only memory cells, as there is no evidence for influenza virus or JHMV replication at sites outside the CNS in these models. A potential efflux of viral Ag during persistence may further be too low to trigger naive T cell activation. In addition, constitutive recruitment of memory cells would lead to an enrichment of virus-specific CD8+ T cells and eventually to CTL exhaustion (2, 5). Neither possibility is supported by experimental data, as splenocytes from JHMV persistently infected mice contain virus-specific CD8+ T cells (32) and persisting influenza virus-specific CTL retain effector function up to 160 days p.i. (11).
The biological significance of persisting CD8+ T
cells at the site of previous infection appears complex. The loss of ex
vivo cytolytic activity by CNS-derived T cells following clearance of
infectious JHMV clearly demonstrates diminished effector function,
in contrast to retention of cytolytic capacity by influenza
virus-specific CD8+ T cells (11).
Based on the differences in tropism and vRNA load during influenza
virus and JHMV infection, the functional CD8+ T
cell discrepancies may reflect Ag-induced anergy within the
JHMV-specific CD8+ T cell population rather than
reduced frequency. Persisting JHMV-specific CD8+
T cells may nevertheless play a role in preventing viral recrudescence
via their ability to secrete IFN-. The total frequency of
IFN--secreting cells was only marginally decreased at day 45
compared with day 8 p.i., despite a decline in the
CD8+ population. This apparent selective
regulation may be beneficial to the host. The early loss of cytolytic
function may minimize tissue damage following perforin-mediated
clearance of infectious virus from microglia and astrocytes (20, 29). However, retention of IFN- secretion during persistence
may be crucial in restraining virus replication in oligodendrocytes.
These cells are refractory to perforin-mediated lysis, but sensitive to
IFN--mediated viral clearance (25). IFN- secretion
in vivo is supported by the continued detection of IFN--encoding
mRNA in the persistently infected CNS (38).
The high frequency of tetramer+ CD8+ T cells within the CNS may reflect regional expansion or efficient recruitment of T cells activated in the CLN. However, the presence of a tetramer- CD8+ T cells during acute JHMV infection suggests nonselective recruitment. Interestingly, the "nonspecific" CD8+ T cell component is retained within the CNS throughout the course of infection. Nonselective lymphocyte recruitment has been demonstrated during the initial phase of CNS inflammation involving antiviral or autoimmune responses (51, 52, 53, 54, 55). Subsequent expansion of distinct T cell subsets within the CNS may result from Ag-driven proliferation (55) or from Ag-mediated retention concomitant with efflux of nonspecific T cells, as shown for CD4+ T cells (51, 52). Similar ratios of tetramer+ vs tetramer- CD8+ T cells within the CNS throughout the course of JHMV infection argue against selective enrichment of virus-specific CD8+ T cells. These data indicate that CD8+ T cells in the CNS may be regulated differently than CD4+ T cells (53), a notion supported by the presence of CTL with no apparent viral specificity during Theiler’s virus infection (56). The identity of the tetramer- CD8+ T cells remains unresolved. Although this population appears to be a nonviral component, responses to epitopes other than the dominant N318 and S510 peptides (33, 34, 36) cannot be ruled out in CB6 F1 mice. One candidate is the Kb-restricted S598 epitope within the viral S protein, identified as subdominant by analysis of CNS-infiltrating cells from mice infected with a neurovirulent JHMV variant (36). However, neither ex vivo CTL or ELISPOT assays confirmed a S598-specific response in CB6 F1 mice (data not shown). In addition to the potential existence of as yet unidentified epitopes, an underestimation of tetramer+ cells due to down-regulation of reactive TCRs may contribute to the apparently nonspecific CD8+ T cell population. The tetramer- CD8+ population may further contain autoantigen-specific T cells, although mice do not show symptoms of autoimmune disease.
The majority of CNS-derived CD8+ T cells,
including the tetramer- subset, were
characterized by CD62Llow, CD44
high, CD11ahigh, and very
late Ag-4 expression, characteristic of activated/memory T cells
accessing the CNS (1, 16, 53, 57). These markers were
unable to distinguish T cells isolated from the acutely or persistently
infected CNS. The most striking phenotypic change during viral
clearance was increased expression of the early activation marker CD69
in both the tetramer+ and
tetramer- subsets. CD69 is rapidly up-regulated
after stimulation in vitro and is thereby implicated as an early marker
of activation (58, 59). However, CD69 did not correlate
with expression of the high affinity IL-2R as a marker for recent
activation and proliferation, consistent with minimal IL-2R
expression on CD8+ T cells during other CNS
infections (45, 60).
CD69+CD8+ T lymphocytes
have been associated with chronic activation (11).
However, CD69 expression may also identify cells in a state of
"anergy post function" (58), and its expression has
been implicated in several immunopathological diseases (59, 61). CD69 expression on CNS-infiltrating
CD8+ T cells may therefore characterize cells
following prolonged Ag contact, resulting in diminished effector
function. This is supported by a high proportion of functionally
unresponsive CD69+CD8+ T
cells in CD4-deficient mice compared with normal control mice
persistently infected with LCMV (6). This is also
consistent with the finding that splenic CD8+ T
cells do not express CD69 during acute peripheral JHMV infection (data
not shown), presumably due to rapid and complete viral clearance.
However, CD69 expression on tetramer-
CD8+ T cells in the present study, in addition to
cytolytic activity of persisting influenza virus-specific
CD69+CD8+ T cells,
indicates that CD69 regulation is only indirectly Ag-driven or
associated with functional down-regulation. Other components in the
local CNS environment, e.g., TNF-, may also play a regulatory role
(62).
An intriguing element of persisting CD8+ T cells is the relationship between biological function, specificity, and clonotypic makeup. Recent evidence from both human and murine models of persistent infections suggests that distinct subsets of primary CTL prevail during memory (6, 12, 13, 32, 63). The inverted ratio of CD8+ T cells responding to two, individually dominant CTL epitopes during the acute and persistent infection demonstrates that immunodominance within the CNS is dynamically regulated during an ongoing response, and thus not strictly dictated by precursor frequencies. The mechanisms responsible for the altered composition of T cell subsets are speculative. Early dominance of N-specific T cells may reflect the large pool of N protein in JHMV-infected cells, giving rise to a high density of Ld-N peptide complexes. The decline of this population may be a consequence of activation-induced cell death or the preferential location of vRNA in the SC at day 12 p.i. However, these factors do not provide a satisfactory explanation for the increased frequency of S-specific T cells, as immunocytochemistry revealed no differences in either the cell type or quantity of N vs S protein distribution. Although both proteins are still present in all cell types at day 12 p.i., expression is limited to oligodendrocytes by day 14 p.i. It is therefore conceivable that oligodendrocytes may favor presentation of the S epitope, as class I Ag processing and presentation may be cell type-specific (13, 64). Changes in cell tropism and thereby class I presentation may provide a mechanism for the retention of responsive "memory" T cell subsets during persistent CNS infection. Furthermore, the existence of cross-reactive CNS epitopes cannot be excluded as a mechanism for preferential maintenance of S-specific T cells (65).
Despite a strong quantitative and qualitative local
CD8+ T cell response, JHMV persists in the CNS in
a noninfectious form. These data demonstrate the retention and/or
ongoing recruitment of activated CD8+ T cells at
the site of infection, which may be attributed to viral persistence, as
evidenced by the switch in immunodominant T cell populations. However,
contributions of the unique environment of the CNS as a target organ
cannot be ruled out. Loss of cytolytic function, but maintenance of
Ag-specific IFN- secretion, further suggests that the CNS has the
propensity to rapidly adjust ongoing immune responses to minimize
tissue destruction. Distinct tissue-specific regulation of
CD8+ T cells is supported by comparative analysis
of T cells derived from the CNS and peripheral tissues during
autoimmune, tumor, viral, or bacterial-induced inflammation (10, 52, 66). As chronic activation of both T cells and
macrophages/microglia have been implicated in pathological conditions
such as autoimmune disease and ongoing primary demyelination, the
extent to which persisting, chronically activated T cells exert
effector function may be larger than previously anticipated and
critical for disease outcome.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cornelia C. Bergmann, Department of Neurology, University of Southern California School of Medicine, 1333 San Pablo Street, MCH 142, Los Angeles, CA 90033. E-mail address:
3 Abbreviations used in this paper: JHMV, JHM strain of mouse hepatitis virus; MHV, mouse hepatitis virus; CLN, cervical lymph nodes; N, nucleocapsid protein; S, spike protein; p.i., postinfection; DBT, delayed brain tumor; SC, spinal cord; vRNA, viral RNA; ELISPOT, enzyme-linked immunospot; APC, allophycocyanin; HPRT, hypoxanthine phosphoriboxyltransferase; LCMV, lymphocytic choriomeningitis virus.
Received for publication May 4, 1999. Accepted for publication June 30, 1999.
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D. D. Sloan, G. Zahariadis, C. M. Posavad, N. T. Pate, S. J. Kussick, and K. R. Jerome CTL Are Inactivated by Herpes Simplex Virus-Infected Cells Expressing a Viral Protein Kinase J. Immunol., December 15, 2003; 171(12): 6733 - 6741. [Abstract] [Full Text] [PDF]
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M. Moniuszko, C. Brown, R. Pal, E. Tryniszewska, W.-P. Tsai, V. M. Hirsch, and G. Franchini High Frequency of Virus-Specific CD8+ T Cells in the Central Nervous System of Macaques Chronically Infected with Simian Immunodeficiency Virus SIVmac251 J. Virol., November 15, 2003; 77(22): 12346 - 12351. [Abstract] [Full Text] [PDF]
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T. S. Kim and S. Perlman Protection Against CTL Escape and Clinical Disease in a Murine Model of Virus Persistence J. Immunol., August 15, 2003; 171(4): 2006 - 2013. [Abstract] [Full Text] [PDF]
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C. Ramakrishna, C. C. Bergmann, R. Atkinson, and S. A. Stohlman Control of Central Nervous System Viral Persistence by Neutralizing Antibody J. Virol., April 15, 2003; 77(8): 4670 - 4678. [Abstract] [Full Text] [PDF]
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E. J. Wherry, J. N. Blattman, K. Murali-Krishna, R. van der Most, and R. Ahmed Viral Persistence Alters CD8 T-Cell Immunodominance and Tissue Distribution and Results in Distinct Stages of Functional Impairment J. Virol., April 15, 2003; 77(8): 4911 - 4927. [Abstract] [Full Text] [PDF]
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M. J. Trifilo, C. C. Bergmann, W. A. Kuziel, and T. E. Lane CC Chemokine Ligand 3 (CCL3) Regulates CD8+-T-Cell Effector Function and Migration following Viral Infection J. Virol., April 1, 2003; 77(7): 4004 - 4014. [Abstract] [Full Text] [PDF]
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C. C. Bergmann, B. Parra, D. R. Hinton, R. Chandran, M. Morrison, and S. A. Stohlman Perforin-Mediated Effector Function Within the Central Nervous System Requires IFN-{gamma}-Mediated MHC Up-Regulation J. Immunol., March 15, 2003; 170(6): 3204 - 3213. [Abstract] [Full Text] [PDF]
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J. Zhou, S. A. Stohlman, D. R. Hinton, and N. W. Marten Neutrophils Promote Mononuclear Cell Infiltration During Viral-Induced Encephalitis J. Immunol., March 15, 2003; 170(6): 3331 - 3336. [Abstract] [Full Text] [PDF]
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N. W. Marten, S. A. Stohlman, J. Zhou, and C. C. Bergmann Kinetics of Virus-Specific CD8+-T-Cell Expansion and Trafficking following Central Nervous System Infection J. Virol., February 15, 2003; 77(4): 2775 - 2778. [Abstract] [Full Text] [PDF]
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 R. G. v. d. Most, K. Murali-Krishna, and R. Ahmed Prolonged presence of effector-memory CD8 T cells in the central nervous system after dengue virus encephalitis Int. Immunol., January 1, 2003; 15(1): 119 - 125. [Abstract] [Full Text] [PDF]
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A. Yu, J. Zhou, N. Marten, C. C. Bergmann, M. Mammolenti, R. B. Levy, and T. R. Malek Efficient Induction of Primary and Secondary T Cell-Dependent Immune Responses In Vivo in the Absence of Functional IL-2 and IL-15 Receptors J. Immunol., January 1, 2003; 170(1): 236 - 242. [Abstract] [Full Text] [PDF]
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A. A. Dandekar and S. Perlman Virus-Induced Demyelination in Nude Mice Is Mediated by {gamma}{delta} T Cells Am. J. Pathol., October 1, 2002; 161(4): 1255 - 1263. [Abstract] [Full Text] [PDF]
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J. Zhou, S. A. Stohlman, R. Atkinson, D. R. Hinton, and N. W. Marten Matrix Metalloproteinase Expression Correlates with Virulence following Neurotropic Mouse Hepatitis Virus Infection J. Virol., June 27, 2002; 76(15): 7374 - 7384. [Abstract] [Full Text] [PDF]
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S.-I. Tschen, C. C. Bergmann, C. Ramakrishna, S. Morales, R. Atkinson, and S. A. Stohlman Recruitment Kinetics and Composition of Antibody-Secreting Cells Within the Central Nervous System Following Viral Encephalomyelitis J. Immunol., March 15, 2002; 168(6): 2922 - 2929. [Abstract] [Full Text] [PDF]
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C. Ramakrishna, S. A. Stohlman, R. D. Atkinson, M. J. Shlomchik, and C. C. Bergmann Mechanisms of Central Nervous System Viral Persistence: the Critical Role of Antibody and B Cells J. Immunol., February 1, 2002; 168(3): 1204 - 1211. [Abstract] [Full Text] [PDF]
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T. Liu, K. M. Khanna, B. N. Carriere, and R. L. Hendricks Gamma Interferon Can Prevent Herpes Simplex Virus Type 1 Reactivation from Latency in Sensory Neurons J. Virol., November 15, 2001; 75(22): 11178 - 11184. [Abstract] [Full Text] [PDF]
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A. E. Matthews, S. R. Weiss, M. J. Shlomchik, L. G. Hannum, J. L. Gombold, and Y. Paterson Antibody Is Required for Clearance of Infectious Murine Hepatitis Virus A59 from the Central Nervous System, But Not the Liver J. Immunol., November 1, 2001; 167(9): 5254 - 5263. [Abstract] [Full Text] [PDF]
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M. T. Liu, H. S. Keirstead, and T. E. Lane Neutralization of the Chemokine CXCL10 Reduces Inflammatory Cell Invasion and Demyelination and Improves Neurological Function in a Viral Model of Multiple Sclerosis J. Immunol., October 1, 2001; 167(7): 4091 - 4097. [Abstract] [Full Text] [PDF]
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C. C. Bergmann, C. Ramakrishna, M. Kornacki, and S. A. Stohlman Impaired T Cell Immunity in B Cell-Deficient Mice Following Viral Central Nervous System Infection J. Immunol., August 1, 2001; 167(3): 1575 - 1583. [Abstract] [Full Text] [PDF]
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R. Holtappels, J. Podlech, N. K. A. Grzimek, D. Thomas, M.-F. Pahl-Seibert, and M. J. Reddehase Experimental Preemptive Immunotherapy of Murine Cytomegalovirus Disease with CD8 T-Cell Lines Specific for ppM83 and pM84, the Two Homologs of Human Cytomegalovirus Tegument Protein ppUL83 (pp65) J. Virol., July 15, 2001; 75(14): 6584 - 6600. [Abstract] [Full Text]
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J. S. Haring, L. L. Pewe, and S. Perlman High-Magnitude, Virus-Specific CD4 T-Cell Response in the Central Nervous System of Coronavirus-Infected Mice J. Virol., March 15, 2001; 75(6): 3043 - 3047. [Abstract] [Full Text]
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P. R. Walker, T. Calzascia, V. Schnuriger, N. Scamuffa, P. Saas, N. de Tribolet, and P.-Y. Dietrich The Brain Parenchyma Is Permissive for Full Antitumor CTL Effector Function, Even in the Absence of CD4 T Cells J. Immunol., September 15, 2000; 165(6): 3128 - 3135. [Abstract] [Full Text] [PDF]
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N. W. Marten, S. A. Stohlman, and C. C. Bergmann Role of Viral Persistence in Retaining CD8+ T Cells within the Central Nervous System J. Virol., September 1, 2000; 74(17): 7903 - 7910. [Abstract] [Full Text]
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G. F. Wu, A. A. Dandekar, L. Pewe, and S. Perlman CD4 and CD8 T Cells Have Redundant But Not Identical Roles in Virus-Induced Demyelination J. Immunol., August 15, 2000; 165(4): 2278 - 2286. [Abstract] [Full Text] [PDF]
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 B. Rehermann Intrahepatic T Cells in Hepatitis B: Viral Control versus Liver Cell Injury J. Exp. Med., April 17, 2000; 191(8): 1263 - 1268. [Full Text] [PDF]
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B. Parra, M. T. Lin, S. A. Stohlman, C. C. Bergmann, R. Atkinson, and D. R. Hinton Contributions of Fas-Fas Ligand Interactions to the Pathogenesis of Mouse Hepatitis Virus in the Central Nervous System J. Virol., March 1, 2000; 74(5): 2447 - 2450. [Abstract] [Full Text]
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